A central question in understanding cell growth concerns the mechanism by which proteins are sorted into the correct compartments. It is believed that protein transport between intracellular compartments is mediated by membranous vesicles and that GTP-binding proteins are involved in the regulation of the directionality and/or accuracy of this vesicular traffic. Our long-term goals are to understand the mechanism of action of the GTP- binding Ypt1 protein in secretion and to identify and study other compartments of the vesicular transport machinery. We plan first to take advantage of the sophisticated classical and molecular genetics of the yeast system to isolate mutations in the Ypt1 gene and identify other genes and gene products with which it interacts. The mutations in the Ypt1 gene and selected genes with which it interacts will be then characterized by cellular and biochemical approaches. The role of the mammalian Ypt1 protein, which shares 70% identify with the yeast protein, will also be investigated by similar means. This proposal is focussed on two specific aims: 1) definition of the different domains of the Ypt1 protein and their function in protein transport, and 2) identification and characterization of other components of the vesicular transport machinery that interact with the Ypt1 protein. The experimental approach will involve generation of mutations in the Ypt1 gene by random and site specific mutagenesis. Genetic, biochemical and physiological analyses of mutations throughout the Ypt1 gene will used to examine the structure-function relationship of the Ypt1 protein and its various activities and interactions. For example, the role of the cycling of Ypt1 protein between GTP and GDP-bound forms in secretion will be examined. Specific functional regions will be determined by fine mapping of ypt1 mutations and identification of sites that are involved in interactions with other proteins. Genetic approaches will be used to detect other genes whose products interact with, or participate in the same pathway as the Ypt1 protein. Approaches similar to those used in the analysis of Ypt1 will be applied to study these other components. Finally, dominant mutations in the Ypt1 gene identified in yeast will be introduced into mammalian cells to investigate their effect on protein transport and growth of these cells. In summary, these studies will help to elucidate the molecular basis of the vesicular transport machinery and the role that GTP-binding proteins play in its regulation. The ability to study both the yeast and the mammalian systems provides a unique opportunity to approach these issues genetically in both systems and to extend the principles learned with yeast to higher cells.